TY - JOUR
T1 - Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy
AU - Stehle, Jochen
AU - Silvers, Robert
AU - Werner, Karla
AU - Chatterjee, Deep
AU - Gande, Santosh
AU - Scholz, Frank
AU - Dutta, Arpana
AU - Wachtveitl, Josef
AU - Klein-Seetharaman, Judith
AU - Schwalbe, Harald
PY - 2014/2/17
Y1 - 2014/2/17
N2 - The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype Gprotein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε- 15N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The metaII and metaIII states emerge in parallel with a relative ratio of about 3:1. Transient formation of the metaIII state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2-loop in the formation of the metaIII state.
AB - The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype Gprotein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε- 15N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The metaII and metaIII states emerge in parallel with a relative ratio of about 3:1. Transient formation of the metaIII state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2-loop in the formation of the metaIII state.
KW - G-protein coupled receptors
KW - NMR spectroscopy
KW - bovine rhodopsin
KW - membrane protein dynamics
KW - photocycles
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U2 - 10.1002/anie.201309581
DO - 10.1002/anie.201309581
M3 - Article
C2 - 24505031
AN - SCOPUS:84893856688
SN - 1433-7851
VL - 53
SP - 2078
EP - 2084
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
IS - 8
ER -